1. Field of the Invention
The present invention relates to an assembly positionable beneath a flexible cell culture substrate membrane that supports the membrane and allows the membrane to stretch across the assembly equally in all directions when vacuum is applied at the periphery of the membrane thereby imparting equibiaxial strain to the growth surface of the membrane and to a cell culture apparatus incorporating the assembly. Alternatively, other geometries of the assembly can be employed that yield uniaxial or gradient strain in the membrane.
2. Prior Art
Elastomeric substrates have been used as surfaces for culturing cells in vitro. These substrates are usually treated to make them hydrophilic and are preferably bonded with matrix proteins. This development arose from the desire to flex cell cultures in vitro instead of culturing cells in a static environment such as exists in conventional cell culture devices. In particular, when cell cultures are grown on membranes which are flexed, the cells are strained and stressed which simulates a dynamic in vivo environment. Stressing of cells causes dramatic morphologic changes and biomechanical responses in the cells which are both long term and short term. Cell culture devices using an elastomeric substrate or a flexible cell culture membrane are flexed or stretched to induce mechanical stress and strain on the cells adhering thereto.
Such flexible cell culture membranes have been incorporated in devices by the Applicant including the single well and multi-well cell culture plates and devices disclosed in U.S. Pat. No. 4,789,601, which is incorporated herein by reference. The wells of this cell culture plate have a floor made from a flexible membrane which is treated to enable cells to adhere and grow thereon. Typically, negative pressure, from a vacuum, is applied to the underside of the cell culture plate to deform downwardly the flexible membranes in the wells of the culture plate.
An improved device is the subject of Applicant""s U.S. Pat. No. 6,048,723, which is incorporated herein by reference. The device disclosed in the ""723 patent is similar to that of the device in the ""601 patent in that it includes a multi-well culture plate having flexible cell culture membranes as the floors of the wells which may be subjected to negative pressure to downwardly flex the membrane. However, the membrane in the device in the ""723 patent is fixed between a base and a body so that either positive or negative pressure may be applied thereto causing upward or downward flexing of the membrane. The pressure differential applied to either of these devices may be released and reapplied to repeatedly apply stress and strain to the cells, thereby mimicking the mechanical load experienced by cells in mechanically active tissues such as the heart, lungs, skeletal muscles, bone, ligament, tendon, cartilage and the like. In this manner, the biological or biochemical responses of cells subjected to a flexed environment may be tested and studied.
However, certain cells normally experience equibiaxial strain in the plane of the cell. By equibiaxial it is meant that the strain is applied equally and simultaneously along both the x-axis and y-axis of the plane of the cell. In some applications of membrane stretching induced by either positive or negative pressure where the membrane is not supported, the cell culture membrane becomes arcuately shaped upon application of a differential pressure to the device. Such arcuate flexing renders difficult an analysis of the biologic responses of the cells since the strain varies in both the circumferential and radial directions. In some cases, the principle strain delivered to cells may be more uniaxial. By uniaxial it is meant that the strain is applied principally along one axis (a strain in one of the x-axis or y-axis). The impact of uniaxial strain in the plane of a cell is likewise important and difficult to analyze.
Accordingly, a need remains for a device for use with a cell culture plate with flexible cell culture membrane wells which provides for well-defined strain to be imposed on cells cultured thereon, wherein the strain is applied equibiaxially, uniaxially or in other defined strain field directions.
This need is met by the loading station assembly of the present invention. The loading station assembly is designed to allow equibiaxial, uniaxial or other directional stretching of a flexible cell culture membrane. In equibiaxial stretching the membrane is maintained in a generally planar configuration while strain is applied equally in both the radial and circumferential directions. In the case of uniaxial stretching, strain is applied in one direction only. The loading station assembly is designed also to prevent deflection of a membrane in a plate otherwise subject to flexation through the use of a special device that does not permit strain to be applied to the membrane. The assembly includes a planar member and at least one post extending from a surface of the planar member. An upper surface of each post is adapted to support the flexible cell culture membrane. The planar member defines at least one aperture and each post is removably received within one of the apertures such that when one of the posts is removed, the membrane is not restricted from flexing downwardly at the position of the absent post. Each post includes a body receivable within one of the apertures and a flange having the upper surface, wherein the flange is positionable against the planar member surface.
The upper surface may be configured to allow equibiaxial, uniaxial or other directional stretching of a membrane. For equibiaxial stretching, the upper surface is preferably circular in shape. For uniaxially stretching, the upper surface preferably has a length which is longer than its width. The upper surface may be formed in a horizontal plane or in a plurality of planes. The planar member also defines a passageway adapted to allow fluid to flow through from one side of the planar member to an opposite side of the planar member such that a pressure differential may be applied to the underside of the membrane.
The present invention further includes a cell culture plate assembly having a cell culture plate defining at least one opening and a flexible cell culture membrane covering the opening. A loading station assembly is positioned adjacent the cell culture plate. The loading station assembly includes a planar member and at least one post extending from a surface of the planar member. An upper surface of each post supports a portion of the flexible membrane covering the opening. The planar member defines at least one aperture and the post is removably received within one of the apertures such that when one of the posts is removed, the cell culture membrane covering an opening is not restricted from flexing downwardly. Each post includes a body receivable within one of the apertures and a flange including the upper surface. The flange is positionable against the planar member. The flange upper surface may be formed in a horizontal plane or in a plurality of planes.
Preferably, each of the flexible member and the loading station assembly is made from a transparent material. In one embodiment of the invention, the post upper surface supports all of the flexible membrane covering the opening. The upper surface may be configured to allow equibiaxial, uniaxial or other directional stretching of the membrane. For equibiaxial stretching, the upper surface of the flange is preferably circular in shape. For uniaxial stretching, the upper surface of the flange preferably has a dimension which is approximately equal to a dimension of the membrane. More preferably, the opening in the cell culture plate is circular and the length of the upper surface is approximately equal to the diameter of the opening and the width of the upper surface is less than the diameter of the opening. A construct, preferably three-dimensional, may be adhered to the membrane to receive cells therein for growth and uniaxial stretching. In this way, a three-dimensional, multi-layered tissue and matrix may be formed. Flexation of the cell-populated construct may enhance the cell and material properties of the forming tissue engineered construct.
The planar member also defines a passageway adapted to allow fluid to flow from one side of the planar member to an opposite side of the planar member.
A complete understanding of the invention will be obtained from the following description when taken in accordance with the accompanying drawing figures wherein like reference characters identify like parts throughout.